摘要
目的研究ADP核糖基化因子6(Arf6)对雄激素非依赖性前列腺癌PC-3细胞株增殖、迁移和侵袭能力的影响并初步探讨其可能的分子作用机制。方法设计合成3条针对不同靶向区域的Arf6特异性si RNA序列,转染细胞后通过real-time PCR和蛋白质印迹法检测其对Arf6的干扰效果,筛选出干扰效果最佳的si RNA序列;通过噻唑盐(MTT)实验、划痕实验、及transwell细胞迁移和侵袭实验观察si RNA干扰Arf6表达对PC-3细胞增殖、迁移和侵袭的影响;蛋白质印迹法检测AKT、p-AKT、ERK1/2、p-ERK1/2和Rac1蛋白表达水平的变化。结果与空白对照组相比,转染阴性对照序列对PC-3细胞内源性Arf6的m RNA和蛋白表达水平无明显影响,3条si RNA序列均能抑制Arf6的表达,其中si RNA-3对PC-3细胞Arf6表达干扰效果最好,Arf6m RNA和蛋白抑制率分别为(91.88±3.13)%和(86.37±0.57)%。si RNA-3干扰Arf6表达抑制PC-3细胞的增殖,且PC-3细胞体外迁移距离和侵袭细胞数较空白和阴性对照组明显减少(P<0.05)。蛋白质印迹法检测发现转染si RNA-3的PC-3细胞p-ERK1/2和Rac1表达水平明显降低,而AKT、p-AKT和ERK1/2表达水平较对照组差异无统计学意义。结论 si RNA干扰Arf6表达可显著抑制PC-3细胞的增殖、迁移和侵袭能力,其分子作用机制可能与p-ERK1/2和Rac1表达下调相关。
Objective To investigate the effects of silencing ADP-ribosylation factor 6（Arf6） on the proliferation, migration, and invasion of prostate cancer cell line PC- 3 and the possible molecular mechanisms. Methods Three Arf6- specific small interfering RNA（si RNA） were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by realtime PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC- 3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2（p-ERK1/2）, ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting. Results Transfection of si RNA- 3 resulted in significantly decreased Arf6 m RNA and protein expression with inhibition rates of（91.88 ± 3.13）% and（86.37 ± 0.57）%, respectively. Arf6 silencing by si RNA- 3 markedly suppressed the proliferation, migration and invasion of PC- 3 cells and reduced the expression levels of p- ERK1/2 and Rac1. Conclusion Silencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC- 3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2016年第6期735-743,共9页
Journal of Southern Medical University
基金
Supported by Natural Science Foundation of Guangdong Province(S2013010014537)
by Science and Technology Planning Project of Guangdong Province(2012B031800263
2014A010107012
2014A020212260)~~
